The purpose of this project is to understand the molecular mechanisms involved in the replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to drastic changes in the host cells structure and metabolic activity. Cellular protein and RNA synthesis are inhibited; the intracellular membrane network becomes rearranged into a network of vesicles that surround and provide a scaffold for viral RNA replication complexes; cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplishes a highly efficient production of viral RNA, proteins, and particles. [unreadable] [unreadable] During FY2008, we have developed and utilized a method to select for sites in picornaviral proteins that are able to tolerate the insertion of extra amino acids while still retaining viability of the viruses that carry these insertions. We have identified such sites so far in four proteins essential for viral RNA replication. We then screened for the abilities of these sites to retain and express viral proteins carrying (a) fluorescent protein tags for localization and live cell imaging studies, (b) epitope tags for identifying cellular and viral protein binding partners, and (c) affinity tags for purification. These viruses encoding tagged proteins are currently being used to determine the location and movement of individual viral proteins in the replication complex, and to determine the functions and interactions of individual viral proteins during infection. [unreadable] [unreadable] Another key viral replication protein has been purified and its biochemical activities and biophysical properties have been analyzed. We have shown that this protein (designated 2C) forms oligomers of 6-8 subunits, and that the oligomeric protein is required for ATPase activity. [unreadable] [unreadable] Finally, we have made great progress in understanding the structure and function(s) of the membrane-associated replication complexes that are induced to form in infected cells. Viral proteins that trigger different aspects of membrane remodeling have been identified, and a cellular protein, guanine nucleotide exchange factor (GBF1) has been shown to participate in the RNA synthesis function of the newly-formed replication complex. This latter finding represents a new cellular pathway in which a host cell factor has been identified on which poliovirus (and other positive-strand RNA viruses) replication depends. Three-dimensional tomographic electron microscopy is in progress to reconstruct the three dimensional stucture of the replication complex membranes.